Strawberry plantlets, regenerated from leaf disks, were used as a model system to study the effect of high concentrations of sugars and dehydration on survival during cryopreservation. After cold acclimation, plantlets imbibed for 3 days (one day each) in 0.5, 0.7 and 1.2 M sucrose and (1.0M sucrose + 0.2M raffinose) and desiccated to 25 % moisture (fwb) in alginate capsules consistently survived cryopreservation. Differential scanning calorimetry revealed only a very small exotherm between -20C and -28C during freezing; a glass transition at -50C and a small melting event at -10C during warming. Conversely, samples with the lowest survival rate, had a large nucleation exotherm at -30C and a devitrification exotherm between -70 and -40C. We conclude that imbibition with sugars, coupled with desiccation treatments, may be used to manipulate freeze tender tissues of strawberry to permit successful cryopreservation.
Manfredo J. Seufferheld and Cecil Stushnoff
Manfredo J. Seufferheld, Cecil Stushnoff, and Philip Forsline
Cryopreservation of mature dormant vegetative buds is a useful method to preserve germplasm of a large number of cold hardy apple cultivars. However, cold tender cultivars have proven to be much more difficult to cryopreserve. Eight cultivars were harvested in September 1993 at Geneva, NY before developing cold hardiness naturally. The twigs were encapsulated with 5% alginate and treated with stepwise imbibition of 0.5 to 1.0 M sucrose. The samples were desiccated over glycerol at 0C. Half of the samples were plunged directly into liquid nitrogen (IN) and the other half were first cooled slowly to -30C. The twigs that had been exposed to prefreezing conditions showed the highest survival (20 to 100%). The samples that were plunged directly in LN survived poorly (0 to 20 %). Samples without encapsulation and no sucrose imbibition had 0% survival. We conclude that this protocol opens up the possibility to expand cryopreservation of cold tender apple cultivars, presently grown only in field gene banks, at great expense and inconvenience.
Manfredo J. Seufferheld, Cecil Stushnoff, John Fitzpatrick, and Philip L. Forsline
Cryopreservation of woody-plant, dormant buds may provide cost-effective, long-term, back-up conservation of germplasm for vegetatively propagated crops that are presently maintained as trees in field gene banks. Dormant buds can be recovered quickly by grafting to dwarfing rootstocks, thus producing flowers for breeding purposes, with minimum potential for inducing somaclonal variants. These attributes are essential to preserve the clonal integrity of unique gene combinations such as those found in tree fruit crops. Previous research has shown that dormant buds from cold-hardy apples can be recovered from storage in liquid nitrogen (LN) with high survival rates (80% to 100%) using controlled desiccation and slow freezing before immersing in LN. On the other hand, dormant buds from cold-tender taxa and buds collected at less than optimal stages for desiccation and freezing have much lower (0% to 50%) survival rates. We increased survival of cold-tender taxa by using a modified vitrification procedure. Dormant apple buds from tender and hardy cultivars were perfused with modified PVS [15% (w/v) ethylene glycol, 15% (w/v) propylene glycol, 7% (w/v) DMSO, and 15% (w/v) glycerol in 0.5 m sorbitol]. Toxicity from the PVS was reduced by dilution soaking in 1 m sorbitol, 0.2 m raffinose, and 15 mm CaCl2 before and after quench-freezing and slow-freezing cryopreservation. Up to 100% of some cold-tender taxa survived. In addition, xylem ray parenchyma tissues that supercool and are normally killed at about -40C with the desiccation protocol survived this vitrification procedure.
Manfredo J. Seufferheld, Cecil Stushnoff, Philip L. Forsline, and Gerardo H. Terrazas Gonzalez
Unlike cold-hardy apple germplasm, dormant vegetative buds from cold-tender accessions require stabilization of meristematic tissue to protect against injury during desiccation and cryopreservation. Dormant buds of six apple cultivars [Malus sylvestris (L.) Mill. var. domestica (Borkh.) Mansf. `Cox's Orange Pippin', `Einshemer', `Golden Delicious', `Jonagold', `K-14', and `Mutsu'] collected at specific intervals in 1993, 1994, and 1995 at Geneva, N.Y., were stabilized by encapsulation in 5% alginate, treated with step-wise imbibition of 0.5 to 1.0 m sucrose and 0.2 m raffinose solution, and desiccated with forced air at 0 °C. Sugar-alginate stabilization reduced injury during desiccation, increased cold-hardiness of the six cold-tender cultivars frozen to -30 °C, and improved recovery following cryopreservation of buds collected before optimal cold acclimation was attained. Sucrose tissue levels did not increase following stabilization treatment, but levels of glucose and fructose, and of an unknown disaccharide increased. This procedure used nontoxic cryoprotectants, and has potential to expand the scope of dormant bud cryopreservation to include cold-tender apple germplasm.